Ca2+ influx though CRAC (Ca2+ release activated Ca2+) channels is critical for immune cell functions. Stimulation through receptors depletes intracellular Ca2+ stores and subsequently leads to opening of CRAC channels on the plasma membrane. Recently, Orai1 (CRACM1) and STIM1 were identified as a pore component of the CRAC channel and a mediator between Ca2+ store depletion and CRAC channel opening, respectively. However, much remains to be understood about the molecular composition and the mechanism of CRAC channel regulation. We have used large scale affinity protein purification under store depleted conditions to identify regulators of the CRAC channel. We have identified CRACR2A (Ca2+ release activated Ca2+ channel Regulator 2A) as a novel regulator of CRAC channels using this approach. The Specific Aims are: (1) To test the hypothesis that Orai1-CRACR2A interaction is critical for the CRAC channel function. The activities of Orai1 mutants incapable of binding to CRACR2A will be analyzed in Orai1-null primary cells. (2) To test the hypothesis that CRACR2A stabilizes the Orai1-STIM1 complex. Our preliminary data suggest that CRACR2A can directly interact with both Orai1 and STIM1. The functions of Orai1 and STIM1 will be examined using CRACR2A knock-down cells. The functional importance of reciprocal interaction of Orai1, STIM1, and CRACR2A will be determined. (3) To test the hypothesis that CRACR2A is critical for the T cell function. Knock-down of CRACR2A in Jurkat T cells decreased Ca2+ entry. We will conditionally target the CRACR2A gene to determine the role of CRACR2A in store-operated Ca2+ entry in peripheral T cells. The experiments on the novel regulator of the CRAC channel are timely because of the very recent discovery of Orai1 and STIM1. In the short term, the proposed experiments should provide new molecular probes for investigating the regulatory mechanism of store-operated Ca2+ entry in mammalian cells, particularly in immune cells. In the long term, we will test the possibility of development of drugs to modulate the function of the CRAC channel; thereby immune activation. Public Health Relevance: We propose to understand the mechanism of a critical step of immune cell activation, calcium entry via plasma membrane calcium channels through proteomic analysis, further functional analysis, and gene manipulation. We successfully indentified a novel molecule, CRACR2A using these approaches and we will focus to elucidate the novel function of this molecule in the immune system. Our study can benefit development of drugs that can activate or repress immune functions as therapy for immune system related problems such as auto-immune diseases or graft rejection.